Neurodegenerative disorders and cancer are major causes of illness and death in the western world. Healing treatment or therapy for neurodegenerative disorders, such as brain stroke, Alzheimer's disease, and Parkinson's disease have eluded the medical and pharmaceutical industries for decades. A key finding is that oxygen-deprived nerve cells produce high levels of glutamate, which stimulate a receptor called an N-Methyl-D-Aspartate (NMDA), known for its ability to cause neuron cell death. Thus, the ability to control the activity of NMDA receptors has become the focus of neuroscience research.
With regard to cancer therapies, one of the major shortcomings of current cancer therapies is the non-selective delivery of the antineoplastic drug to both targeted tumor cells and healthy cells. Enhanced selectivity of such drugs could diminish their associated toxicity by reducing their uptake by healthy cells. Moreover, selective delivery would increase drug potency by lowering the effective dosage required to kill the affected cell type. Vectored systems, which have enhanced affinity for cancer cells would be an important advance in cancer therapy.
Polyamines are naturally occurring amines, which form polycations in vivo. These stabilize DNA architectures and are cellular growth factors. All cells contain some form of the native polyamines: putrescine, spermidine or spermine. Rapidly dividing cells (such as cancer cells) require large amounts of polyamines, and cells can either biosynthesize or import these essential growth factors. Many tumor cell lines have been shown to have very high levels of polyamines and an active polyamine transporter.
Polyamine structures have been exploited for use in various drug strategies, such as demonstrated in U.S. Pat. Nos. Bergeron, 6,342,534 and 5,866,613; Prakash, 5,109,024; Iwata 6,319,956 and Published application 2002/0067472 A1; Bowlin 5,719,193; and, Klosel 6,281,371 B1 and published document “A Comparison of Structure-Activity Relationships between Spermidine and Spermine Analogue Antineoplastics,” by Bergeron, R. J.; Feng, Y.; Weimar, W. R.; McManis, J. S.; Dimova, H.; Porter, C.; Raisler, B.; Phanstiel IV, O. J. Med. Chem. 1997, 40, No. 10, 1475-1494.
Bergeron U.S. Pat. No. 6,342,534 should be considered with respect to Column 3, lines 51-67 and Column 4, lines 39-48, Table 1 and Table 2, in which the emphasis is on bis-substituted tetraamine systems terminated with N-ethyl, N-piperidinyl, and pyridinyl units. Bowlin referred to above should be considered with respect to Column 1, lines 51-67 in which is described compounds useful for potentiating the cellular immune response. Bowlin's compounds are limited to activating cells to be killed by the immune system. Thus, Bowlin requires an immune system to work with their disclosed drugs.
Other research in using polyamine conjugates for cellular entry has been described in published documents (Cohen, G. M.; Cullis, P.; Hartley, J. A.; Mather, A. Symons, M. C. R.; Wheelhouse, R. T. Targeting of Cytotoxic Agents by Polyamines: Synthesis of a Chloroambucil-Spermidine Conjugate. J. Chem. Soc. Chem. Commun. 1992, 298-300; Cullis, P. M.; Merson-Davies, L.; Weaver, R. Conjugation of a polyamine to the bifunctional alkylating agent chlorambucil does not alter the preferred cross-linking site in duplex DNA. J. Am. Chem. Soc. 1995, 117, 8033-8034; Phanstiel IV, O.; Price, H. L; Wang, L.; Juusola, J.; Kline, M.; Shah, S. M. The Effect of Polyamine Homologation on the Transport and Cytotoxicity Properties of Polyamine-(DNA-Intercalator) Conjugates. J. Org. Chem. 2000, 65, 5590-5599; Wang, L.; Price, H. L.; Juusola, J.; Kline, M.; Phanstiel, IV, O. “Influence of Polyamine Architecture on the Transport and Topoisomerase II Inhibitory Properties of Polyamine DNA-Intercalator Conjugates,” J. Med. Chem. 2001, 44, 3682-3691; Delcros, J-G.; Tomasi, S.; Carrington, S.; Martin, B.; Renault, J.; Blagbrough, I. S.; Uriac, P. Effect of spermine conjugation on the cytotoxicity and cellular transport of acridine. J. Med. Chem., 2002, 45, 5098-5111; “Synthesis and Biological Evaluation of N1-(anthracen-9-ylmethyl)triamines as Molecular Recognition Elements for the Polyamine Transporter,” Wang, C.; Delcros, J-G.; Biggerstaff, J.; Phanstiel IV, O. J. Med. Chem., 2003, 46, 2663-2671; “Molecular Requirements for Targeting the Polyamine Transport System: Synthesis and Biological Evaluation of Polyamine-Anthracene Conjugates,” Wang, C.; Delcros, J-G.; Biggerstaff, J.; Phanstiel IV, O. J. Med. Chem. 2003, 46, 3672-2682; “Defining the Molecular Requirements for the Selective Delivery of Polyamine-Conjugates into Cells Containing Active Polyamine Transporters,” Wang, C.; Delcros, J-G.; Cannon, L.; Konate, F.; Carias, H.; Biggerstaff, J.; Gardner, R. A.; Phanstiel IV, O. J. Med. Chem. 2003, 46, 5129-5138; “N1-Substituent Effects in the Selective Delivery of Polyamine-Conjugates into Cells Containing Active Polyamine Transporters”Gardner, R. A.; Delcros, J-G.; Koriate, F.; Breitbeil III, F.; Martin, B.; Sigman, M.; Huang, M.; Phanstiel IV, O. J. Med. Chem. 2004, 47, 6055-6069.)
The prior art by Cullis et al is limited to delivering a DNA-alkylating agent (chlorambucil) to cells using spermidine, a non-optimal polyamine vector. The chlorambucil substituent is linked via a tether to the internal N4-nitrogen of the spermidine chain. Recent findings have shown this internal N-alkylation motif used by Cullis to be a less than optimal arrangement for using the polyamine transporter. The previous publications by Phanstiel IV et al are limited to branched polyamine systems built from spermine and spermidine platforms, again using non-optimized polyamine vectors. The report by Blagbrough et al focused on using tetraamine derivatives of spermine to deliver acridine to cells. Blagbrough's compounds are limited by the use of less than optimal spermine vectors to deliver a less potent acridine drug into cells.
The more recent Phanstiel IV papers (2003-2004) illustrate this technology with linear triamines and tetraamine systems in targeting cancer cells via the polyamine transporter.
Since the 1980s several laboratories have probed the transport properties of polyamines into various cell types (E. coli, yeast and mammals). The polyamine transporter in E. coli is perhaps the best understood as the transporter gene and several protein gene products (Pot A-F) have been identified. In particular the PotB and PotC proteins form a trans-membrane channel, which facilitates polyamine transport. PotD is a periplasmic, polyamine-binding protein, which prefers spermidine over putrescine. Moreover, the X-ray crystal structure of spermidine bound to PotD revealed that the molecular recognition events involved in spermidine binding is controlled by specific amino acid residues and a bound water molecule. Specifically, through this water molecule, the bound spermidine molecule forms two hydrogen bonds with Thr 35 and Ser 211. In a related study the PotF protein was shown to selectively bind putrescine. The PotF crystal structure, in combination with the mutational analysis, revealed the residues crucial for putrescine binding (Trp-37, Ser-85, Glu-185, Trp-244, Asp-247, and Asp-278) and the importance of water molecules for putrescine recognition. Therefore, the E. coli studies provided a striking example of how cells can discriminate between structurally similar di- and tri-amine substrates, (e.g., putrescine (PUT) and spermidine (SPD), respectively). While significant work has also been accomplished in yeast and other systems, the proteins involved in mammalian polyamine transport have not yet been isolated and characterized beyond a kinetic description. Clearly, the lack of structural detail associated with the mammalian polyamine transporter is a glaring void in the knowledge base.
The NMDA receptor is known to have a polyamine binding site, which modulates its action. Moreover, it is known that the site(s) responsible for both the agonist and antagonist activity of polyamine derivatives reside in a single subunit of the NMDA receptor-channel complex (NR2). This phenomenon has been reported in Ransom, R. W.; Stec, N. L.; Cooperative Modulation of [3H]MK-801 binding to the n-methyl-D-Aspartate Receptor ion Channel by Glutamate, Glycine and Polyamines. J. Neurochem. 1988, 51, 830-836 and in Williams, K.; Romano, C.; Molinoff, P. B. Effects of Polyamines on the binding of [3H] MK-801 to the N-methyl-D-Aspartate receptor: Pharmacological Evidence for the Existence of a Polyamine Recognition Site. Mol. Pharmacol. 1989, 36, 575-581 and Williams, K.; Zappia, A. M.; Pritchett, D. B.; Shen, Y. M.; Molinoff, P. B. Sensitivity of the N-Methyl-D-Aspartate receptor to polyamines is Controlled by NR2 Subunits. Mol. Pharmacol. 1994, 45, 803-809. In 1995, Bergeron et al. discussed multiple uses of polyamines in the Journal of Medicinal Chemistry 1995, 38, 425-442, “Impact of Polyamine Analogues on the NMDA Receptor.” In addition to antineoplastic activity against tumor cells, N-terminally dialkylated tetraamines were reported to have a potent effect on neuromuscular activity in the gut, function in modulating neural transmission and exhibit a pronounced biphasic action on NMDA receptor function. What was not known is the optimal polyamine architecture to selectively inhibit the NR2 subunit of the NMDA receptor, a site responsible for neuronal cell death. A success in this area would provide the medical community with a new tool and potential therapy for the treatment of stroke and neurodegenerative diseases.
Indeed, very selective and effective tetraamine derivatives for treatment of neurodegenerative disorders, such as stroke, Alzheimer's disease, Parkinson's disease and the like would satisfy a very significant commercial demand in the medical and pharmaceutical industries.